title: Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels.
abstract: Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca(2+)/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca(2+)/calmodulin, one at submicromolar Ca(2+) concentrations and one in the micromolar Ca(2+) range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca(2+)/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca(2+) signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca(2+) regulation in anoctamin Cl(-) channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types.
authors.affiliation: Department of Molecular Physiology, Centre for Organismal Studies, Heidelberg University, 69120 Heidelberg, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Vocke
authors.forename: Kristin
authors.initials: K
authors.lastname: Dauner
authors.forename: Anne
authors.initials: A
authors.lastname: Hahn
authors.forename: Anne
authors.initials: A
authors.lastname: Ulbrich
authors.forename: Jana
authors.initials: J
authors.lastname: Broecker
authors.forename: Sandro
authors.initials: S
authors.lastname: Keller
authors.forename: Stephan
authors.initials: S
authors.lastname: Frings
authors.forename: Frank
authors.initials: F
authors.lastname: Möhrlen
mesh_terms: D000200:Action Potentials; D000595:Amino Acid Sequence; D000818:Animals; D000075369:Anoctamin-1; D000075368:Anoctamins; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009474:Neurons / Q000378:metabolism / Q000502:physiology; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012160:Retina / Q000378:metabolism
chemical_list: C552942:ANO1 protein, rat; C575623:Ano2 protein, rat; D000075369:Anoctamin-1; D000075368:Anoctamins; D002147:Calmodulin; D018118:Chloride Channels; D020033:Protein Isoforms; D002118:Calcium